The major objective of our research program is the development of an in-depth understanding of many aspects of superhelical DNA in relation to biological function. In particular we have focused on obtaining insight into the nature of enhanced transcriptional activity produced by supercoiling. We have developed the capacity to modify superhelical DNA with a water-soluble carbodiimide N-cyclohexyl-N'-beta-(4,methylmorpholium) ethyl carbodiimide (CMC) and have shown that modification rapidly diminished transcription. Using short reaction times it is possible to limit CMC modification to 10.7 CMC/SV40 DNA which represents approximately one CMC per E. coli RNA polymerase promoter or binding site. The accumulated data reveals that promoters are highly preferential targets for modification in superhelical SV40 DNA. It is of considerable interest to extend this work in order to elucidate the mechanism of CMC inhibition of polymerase binding as well as to test whether this behavior occurs as a general phenomenon. In order to accomplish this we have turned our attention as well to the in vitro characterization of the transcription of Colicin E1 DNA and other plasmids. The following specific aims are envisioned. a) Localization of CMC in relationship to promoter sites in SV40 and other superhelical DNAs. In particular Co1E1 and pBR322 can serve as an excellent model system. b) Analysis of CMC modification as a function of superhelix density and other solvent and physical parameters. c) Chemical and physical characterization of promoters in relaxed and superhelical DNA in regard to RNA polymerase binding. d) Development of a radioimmune assay for the CMC modified DNA. e) Develop a new procedure for direct sequencing of superhelical DNA with special attention to modified DNA. f) Utilization of abortive initiation to evaluate the binding and rate parameters for promoters on superhelical DNA.